Showing papers on "Pregnenolone published in 2008"
TL;DR: It is shown that TRPM3, a divalent-permeable cation channel, is rapidly and reversibly activated by extracellular pregnenolone sulphate, a neuroactive steroid, enabling unanticipated crosstalk between steroidal and insulin-signalling endocrine systems.
Abstract: Transient receptor potential (TRP) cation channels are renowned for their ability to sense diverse chemical stimuli. Still, for many members of this large and heterogeneous protein family it is unclear how their activity is regulated and whether they are influenced by endogenous substances. On the other hand, steroidal compounds are increasingly recognized to have rapid effects on membrane surface receptors that often have not been identified at the molecular level. We show here that TRPM3, a divalent-permeable cation channel, is rapidly and reversibly activated by extracellular pregnenolone sulphate, a neuroactive steroid. We show that pregnenolone sulphate activates endogenous TRPM3 channels in insulin-producing beta cells. Application of pregnenolone sulphate led to a rapid calcium influx and enhanced insulin secretion from pancreatic islets. Our results establish that TRPM3 is an essential component of an ionotropic steroid receptor enabling unanticipated crosstalk between steroidal and insulin-signalling endocrine systems.
321 citations
TL;DR: Observations suggest that DHP and P (i.e. two neuroactive steroids interacting with progesterone receptor) may be considered protective agents in case of nerve crush injury.
110 citations
TL;DR: The purpose of this study was to assess the reasons for discrepancies in reports of induction of some cytochrome P450 enzymes and ABC transporters, and use a standardized approach to compare activators of NRs.
Abstract: Background and purpose:
Certain nuclear receptors (NRs) such as the constitutive androstane receptor (CAR), pregnane X receptor (PXR) and farnesoid X receptor (FXR) mediate induction of some cytochrome P450 enzymes and ABC transporters but conflicting reports exist. The purpose of this study was to assess the reasons for these discrepancies and use a standardized approach to compare activators of NRs.
Experimental approach:
Dexamethasone, pregnenolone 16α-carbonitrile, rifampicin, phenobarbital and chenodeoxycholic acid were incubated with HepG2, Caco-2 and cryopreserved human hepatocytes prior to analysis of mRNA and protein for CYP2B6, CYP3A4, CYP3A5, ABCB1, ABCC1, ABCC2, PXR, CAR and FXR.
Key results:
Dexamethasone significantly up-regulated PXR, CYP3A4 and ABCB1 expression in HepG2 and Caco-2 cells. As a result, including dexamethasone as a media supplement masked the induction of these genes by pregnenolone 16α-carbonitrile, which may explain discrepancies between previous reports. In the absence of dexamethasone, significant activator-dependent induction was observed in all cell types. Significant correlations were observed between the fold increase in mRNA and in protein, which were, for most instances, logarithmic. Changes in mRNA expression were greater in cell lines than primary cells but for most transcripts correlations were observed between fold increases in HepG2 and hepatocytes.
Conclusions and implications:
Clearly, no in vitro system can imitate the physiology of a hepatocyte or intestinal cell within an intact organ in vivo, but these data explain some of the discrepancies reported between laboratories and have important implications for study design.
British Journal of Pharmacology (2008) 153, 805–819; doi:10.1038/sj.bjp.0707601; published online 26 November 2007
110 citations
TL;DR: It is demonstrated that the LC-MS/MS method allows the assessment of neuroactive steroids in the nervous system with high sensitivity and specificity and that diabetes strongly affects their levels, providing a further basis for new therapeutic tools based on neuro active steroids aimed at counteracting diabetic neuropathy.
102 citations
TL;DR: Although available direct analytical methods have failed to detect levels of PREGS above 0.1-0.3 ng/g in brain tissue, it may be premature to completely exclude the local formation of biologically active PREGS within specific and limited compartments of the nervous system.
97 citations
TL;DR: In addition to their effect as direct ER agonists, the effects of endocrine disruptors may be evaluated and interpreted as interference with steroidogenesis and with the steroidal regulation of the normal development and function of juvenile, male and female individuals.
Abstract: Biologically active steroids are synthesised de novo in specialised cells of several organs, including the adrenal gland, testis, ovary, brain, placenta and adipose tissue. Regardless of organ or tissue, the rate-limiting step in steroid hormone synthesis is the movement of cholesterol across the mitochondrial membrane (i.e. from the outer to the inner membrane) mediated by the steroidogenic acute regulatory (StAR) protein. Subsequent conversion of cholesterol to pregnenolone by cytochrome P450 side-chain cleavage (P450scc) represents the initiation of steroidogenesis. Chemically mediated disruption of StAR and P450scc expression may represent the first step in the sequence of related event cascades underlying xenoestrogen-induced toxicity and transmittable disturbances to the whole organism level. This may include, but is not limited to, alterations in sexual differentiation, growth, reproduction, development and metabolism. Despite the integral role of StAR and P450scc in acute steroidogenesis, and popular demand from regulatory agencies, bioassays for evaluating the effect of endocrine-disrupting chemicals have the potential to overlook chemicals that may modulate estrogenic responses through mechanisms that do not involve direct binding to estrogen receptors (ERs). In addition to their effect as direct ER agonists, the effects of endocrine disruptors may be evaluated and interpreted as interference with steroidogenesis and with the steroidal regulation of the normal development and function of juvenile, male and female individuals. Knowledge of these effects is scarce, indicating that relatively little is known about the mechanisms or mode-of-action of chemical alterations to steroidogenesis and their potential toxicity for wildlife species. In addition, analytical methods for the complete adaptation of these responses as biomarkers of response and effect are yet to be properly validated.
93 citations
TL;DR: Data obtained are shown that endogenous steroids synthesized in the SC are involved in the modulation of nociceptive mechanisms and the physiological relevance of the modification of endogenous steroid formation in theSC during painful situation was discussed.
76 citations
TL;DR: The results of this study suggested that the novel N,N-disubstituted indol-3-ylglyoxylamides may represent a promising class of compounds potentially suited for the treatment of anxiety disorders.
Abstract: Novel N,N-disubstituted indol-3-ylglyoxylamides (1−56), bearing different combinations of substituents R1−R5, were synthesized and evaluated as ligands of the translocator protein (TSPO), the 18 kDa protein representing the minimal functional unit of the “peripheral-type benzodiazepine receptor” (PBR). Most of the new compounds showed a nanomolar/subnanomolar affinity for TSPO and stimulated steroid biosynthesis in rat C6 glioma cells with a potency similar to or higher than that of classic TSPO ligands such as PK 11195. Moreover, when evaluated in vivo by means of the elevated-plus-maze (EPM) paradigm in the rat, compound 32, the best-performing derivative in terms of TSPO affinity and pregnenolone production, showed clear anxiolytic effects. The results of this study suggested that the novel N,N-disubstituted indol-3-ylglyoxylamides may represent a promising class of compounds potentially suited for the treatment of anxiety disorders.
75 citations
TL;DR: Findings indicate that CSF DHEA and pregnenolone levels are correlated with temporal cortex brain levels of these neurosteroids and thatCSF D HEA is elevated in AD and related to neuropathological disease stage.
Abstract: Objective: It is currently unknown whether cerebrospinal fluid (CSF) neurosteroid levels are related to brain neurosteroid levels in humans. CSF and brain dehydroepiandrosterone (DHEA) levels are elevated in patients with Alzheimer’s disease (AD), but it is unclear whether CSF DHEA levels are correlated with brain DHEA levels within the same subject cohort. We therefore determined DHEA and pregnenolone levels in AD patients (n = 25) and cognitively intact control subjects (n = 16) in both CSF and temporal cortex. Design: DHEA and pregnenolone levels were determined by gas chromatography/mass spectrometry preceded by HPLC. Frozen CSF and temporal cortex specimens were provided by the Alzheimer’s Disease Research Center at Duke University Medical Center. Data were analyzed by Mann-Whitney U test statistic and Spearman correlational analyses. Results: CSF DHEA levels are positively correlated with temporal cortex DHEA levels (r = 0.59, P < 0.0001) and neuropathological disease stage (Braak and Braak) (r = 0....
67 citations
TL;DR: While chrysin itself is unable to induce StAR gene expression and steroidogenesis, it appears to function by increasing the sensitivity of Leydig cells to cAMP stimulation.
Abstract: During the aging process of males, testosterone biosynthesis declines in testicular Leydig cells resulting in decreases in various physiological functions. To explore the possibility of delaying the decline using food supplements, we have studied steroidogenic effects of a natural flavonoid, chrysin, in mouse Leydig cells. Chrysin dramatically increased cyclic AMP (cAMP)-induced steroidogenesis in MA-10 mouse Leydig tumor cells. This result was confirmed using Leydig cells isolated from mouse testes. The steroidogenic effect of chrysin is not associated with an increase in expression of the P450 side-chain cleavage enzyme, required for the conversion of cholesterol to pregnenolone. In addition, when 22(R)hydroxylcholesterol was used as a substrate, chrysin induced a non-significant increase in steroid hormone, suggesting that the majority of the observed increase in steroidogenesis was due to the increased supply of substrate cholesterol. These observations were corroborated by showing that chrysin induced a marked increase in the expression of steroidogenic acute regulatory (StAR) protein, the factor that controls mitochondrial cholesterol transfer. Also, chrysin significantly increased StAR promoter activity and StAR mRNA level. Further studies indicated that this compound depressed expression of DAX-1, a repressor in StAR gene transcription. In the absence of cAMP, chrysin did not increase steroidogenesis. However, when a sub-threshold level of cAMP was used, StAR protein and steroid hormone were increased by chrysin to the levels seen with maximal stimulation of cAMP. These results suggest that while chrysin itself is unable to induce StAR gene expression and steroidogenesis, it appears to function by increasing the sensitivity of Leydig cells to cAMP stimulation.
63 citations
TL;DR: Investigations of the time evolution of reaction progress have indicated that the substrates stimulate activity of BVMO(s) of P. lilacinum AM111, which is able to process 3beta-hydroxy-5-ene steroid substrates.
TL;DR: It is indicated that TGFB1 counteracts stimulation of E(2) and P(4) synthesis in granulosa cells by inhibiting key enzymes involved in the conversion of androgens to E-2 and cholesterol to P-4 without shutting down HSD17B reducing activity and HSD3B activity.
Abstract: Survival and inhibitory factors regulate steroidogenesis and determine the fate of developing follicles. The objective of this study was to determine the role of transforming growth factor-beta1 (TGFB1) in the regulation of estradiol-17beta (E(2)) and progesterone (P(4)) secretion in FSH-stimulated bovine granulosa cells. Granulosa cells were obtained from 2 to 5 mm follicles and cultured in serum-free medium. FSH dose (1 and 10 ng/ml for 6 days) and time in culture (2, 4, and 6 days with 1 ng/ml FSH) increased E(2) secretion and mRNA expression of E(2)-related enzymes cytochrome P450 aromatase (CYP19A1) and 17beta-hydroxysteroid dehydrogenase type 1 (HSD17B1), but not HSD17B7. TGFB1 in the presence of FSH (1 ng/ml) inhibited E(2) secretion, and decreased mRNA expression of FSH receptor (FSHR), CYP19A1, and HSD17B1, but not HSD17B7. FSH dose did not affect P(4) secretion and mRNA expression of 3beta-hydroxysteroid dehydrogenase (HSD3B) and alpha-glutathione S-transferase (GSTA), but inhibited the amount of steroidogenic acute regulatory protein (STAR) mRNA. Conversely, P(4) and mRNA expression of STAR, cytochrome P450 side-chain cleavage (CYP11A1), HSD3B, and GSTA increased with time in culture. TGFB1 inhibited P(4) secretion and decreased mRNA expression of STAR, CYP11A1, HSD3B, and GSTA. TGFB1 modified the formation of granulosa cell clumps and reduced total cell protein. Finally, TGFB1 decreased conversion of androgens to E(2), but did not decrease the conversion of estrone (E(1)) to E(2) and pregnenolone to P(4). Overall, these results indicate that TGFB1 counteracts stimulation of E(2) and P(4) synthesis in granulosa cells by inhibiting key enzymes involved in the conversion of androgens to E(2) and cholesterol to P(4) without shutting down HSD17B reducing activity and HSD3B activity.
TL;DR: Data indicate that the endozepine ODN, released by glial cell processes in the vicinity of 3β‐hydroxysteroid dehydrogenase‐containing neurones, stimulates the biosynthesis of neurosteroids through activation of central‐type benzodiazepines receptors.
Abstract: Neurosteroids may play a major role in the regulation of various neurophysiological and behavioural processes. However, while the biochemical pathways involved in the synthesis of neuroactive steroids in the central nervous system are now elucidated, the mechanisms controlling the activity of neurosteroid-producing cells remain almost completely unknown. In the present study, we have investigated the effect of the octadecaneuropeptide (ODN), an endogenous ligand of benzodiazepine receptors, in the control of steroid biosynthesis in the frog hypothalamus. Glial cells containing ODN-like immunoreactivity were found to send their thick processes in the close vicinity of neurones expressing the steroidogenic enzyme 3β-hydroxysteroid dehydrogenase. Exposure of frog hypothalamic explants to graded concentrations of ODN (10−10−10−5m) produced a dose-dependent increase in the conversion of tritiated pregnenolone into various radioactive steroids, including 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone and dihydrotestosterone. The ODN-induced stimulation of neurosteroid biosynthesis was mimicked by the central-type benzodiazepine receptor (CBR) inverse agonists methyl β-carboline-3-carboxylate (β-CCM) and methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM). The stimulatory effects of ODN, β-CCM and DMCM on steroid formation was markedly reduced by the CBR antagonist flumazenil. The ODN-evoked stimulation of neurosteroid production was also significantly attenuated by GABA. Collectively, these data indicate that the endozepine ODN, released by glial cell processes in the vicinity of 3β-hydroxysteroid dehydrogenase-containing neurones, stimulates the biosynthesis of neurosteroids through activation of central-type benzodiazepines receptors.
TL;DR: Increased brain levels of neurosteroids with positive allosteric modulatory actions at the neuronal GABA‐A receptor offer a cogent explanation for the notion of ‘increased GABAergic tone’ in HE.
Abstract: Hepatic encephalopathy (HE) is a neuropsychiatric complication of both acute and chronic liver failure characterized by progressive neuronal inhibition. Some neurosteroids are potent positive allosteric modulators of the gamma-aminobutyric acid (GABA)-A receptor complex, and 'increased GABAergic tone' has been proposed to explain the neuroinhibition characteristics of HE. Brain levels of the neurosteroids pregnenolone, allopregnanolone and tetrahydrodesoxycorticosterone (THDOC) and the functional status of the GABA-A receptor complex were assessed in rats following portacaval anastomosis (PCA). Effects of indomethacin, an inhibitor of the 3alpha-hydroxysteroid dehydrogenase enzyme involved in neurosteroid synthesis, on PCA rat locomotor activity and brain neurosteroid levels were also assessed. Significant increases of the neurosteroid pregnenolone (2.6-fold), allopregnanolone (1.7-fold) and THDOC (4.7-fold) were observed in brains of PCA rats. Brain levels of these neurosteroids were in the nanomolar range, sufficient to exert positive allosteric modulatory effects at the GABA-A receptor. Indomethacin (0.1-5 mg kg(-1)) ameliorated dose-dependently the locomotor deficit of PCA rats and concomitantly normalized brain levels of allopregnanolone and THDOC. Increased brain levels of neurosteroids with positive allosteric modulatory actions at the neuronal GABA-A receptor offer a cogent explanation for the notion of 'increased GABAergic tone' in HE. Pharmacological approaches using agents that either reduce neurosteroid synthesis or modulate the neurosteroid site on GABA-A receptor could offer new therapeutic tools for the management and treatment of HE.
TL;DR: A role for the central σ receptors particularly sigma-1 (σ1) receptors in the anti-depressant-like effects of neurosteroids is suggested, and also the possible involvement of sigma (σ) receptors.
Abstract: The present study investigated the effects of neurosteroids dehydroepiandrosterone sulfate (DHEAS) or pregnenolone sulfate (PS) on the tail-suspension test (TST) of depression in mice, and also the possible involvement of sigma (sigma) receptors. Immobility time in the TST was measured for a total period of 6 min. DHEAS (10 and 40 mg/kg, s.c.) or PS (40 mg/kg, s.c.) significantly reduced the immobility period without accompanying changes in the locomotor activity in mice. The effect on behavioural despair by DHEAS (10 and 40 mg/kg, s.c.) and PS (40 mg/kg, s.c.) was blocked by BD 1047 (1 mg/kg, s.c.), a novel sigma1-receptor antagonist, progesterone (10 mg/kg, s.c.), a sigma-receptor antagonistic neurosteroid or rimcazole (5 mg/kg, s.c.), another sigma1-receptor antagonistic property, respectively. The treatments and their combination did not alter the motor activity in mice. These data suggested a role for the central sigma receptors particularly sigma-1 (sigma1) receptors in the anti-depressant-like effects of neurosteroids.
TL;DR: A selective and amino-acid sequence-dependent action of A beta on neurosteroidogenesis is demonstrated, which may explain A beta 1-42 ability to exert either protective or deleterious effects on nerve cells.
Journal Article•
TL;DR: The results of the present in vitro studies suggest that excitatory neurosteroids DHEA, DHEAS and PGL at physiological concentrations participate in the inhibition of cortical neuronal degeneration elicited by staurosporine and glutamate, whereas the most potent positive modulator of GABA(A) receptor--Allo--has no effect.
Abstract: Some neurosteroids show neuroprotective action in in vitro and in vivo studies, but their interaction with apoptotic/necrotic processes has been only partially unraveled. The aim of the present study was to examine the effect of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), pregnenolone (PGL) and allopregnanolone (Allo) on staurosporine-, glutamate-, and NMDA-induced damage in primary cortical neuronal culture. DHEA, DHEAS and PGL (0.1 and 1 microM) inhibited the staurosporine-evoked LDH release and decreased the number of apoptotic cells as shown by Hoechst;s staining, whereas Allo was without effect. The neurosteroids affected neither the staurosporine-evoked changes in caspase-3 activity nor the decrease in mitochondrial membrane potential. It was also shown that protective effects of DHEA, DHEAS and PGL against staurosporine-induced LDH release were attenuated by extracellular signal-regulated kinase (ERK)--mitogen-activated protein kinase (MAPK) inhibitor--PD 98059 (5 microM) but not by phosphatidylinositol-3-kinase (PI3-K) inhibitors such as LY 294002 (1 microM) or wortmannin (10 nM). The involvement of ERK2-MAPK in protective effects of neurosteroids was confirmed by Western blot study. Further study demonstrated that glutamate-induced cell damage was attenuated by DHEA, DHEAS, and PGL, but not by Allo. None of the steroids influenced NMDA-induced LDH release. The results of the present in vitro studies suggest that excitatory neurosteroids DHEA, DHEAS and PGL at physiological concentrations participate in the inhibition of cortical neuronal degeneration elicited by staurosporine and glutamate, whereas the most potent positive modulator of GABA(A) receptor--Allo--has no effect. Moreover, neurosteroids appear to attenuate the staurosporine-induced cell damage in a caspase-3 independent way and their neuroprotective mechanism of action involves the increase in ERK-MAPK phosphorylation.
TL;DR: Data indicate that neurosteroids prevent SH‐SY5Y cell damage related to oxidative processes and activation of mitochondrial apoptotic pathway, and neuroprotective effects of DHEA, DHEAS seem to depend on PI3‐K and ERK/MAPK signaling pathways.
Abstract: Neurosteroids are important regulators of central nervous system function and may be involved in processes of neuronal cell survival. This study was undertaken to test the effect of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), pregnenolone (PGL), pregnenolone sulfate (PGLS), and allopregnanolone (Allo) on hydrogen peroxide- and staurosporine-induced toxicity in SH-SY5Y cells. It has been found that DHEAS inhibited the hydrogen peroxide toxicity in a concentration-dependent manner, whereas DHEA was active only at higher doses. PGL and PGLS showed neuroprotective effects only at the lowest concentration. Allo had no significant effect on hydrogen peroxide-evoked lactate dehydrogenase release and at the highest concentration aggravated its toxic effects. Next part of this study evaluated neurosteroid effects on staurosporine-induced apoptosis. DHEAS, DHEA, and PGL significantly antagonized effects of staurosporine on both caspase-3 activity and mitochondrial membrane potential. PGLS and Allo inhibited the staurosporine-induced changes in both apoptotic parameters only at the lowest concentration. Antiapoptotic properties of neurosteroids were positively verified by Hoechst staining. Furthermore, as shown by calcein assay, DHEA, DHEAS, and PGL increased viability of staurosporine-treated cells, and these effects were attenuated by specific inhibitors of phosphatidylinositol 3-kinase (PI3-K) and extracellular signal-regulated protein kinase (ERK)-mitogen activated protein kinase (MAPK). These data indicate that neurosteroids prevent SH-SY5Y cell damage related to oxidative processes and activation of mitochondrial apoptotic pathway. Moreover, neuroprotective effects of DHEA, DHEAS seem to depend on PI3-K and ERK/MAPK signaling pathways. It can be suggested that, at physiological concentrations, all studied neurosteroids participate in the inhibition of neuronal apoptosis, but with various potencies. © 2008 Wiley-Liss, Inc.
TL;DR: A surprising functional association between TRPM3, a mysterious member of the family of transient receptor potential (TRP) cation channels, and the sulphated version of pregnenolone, 'mother' of all steroid hormones, has been identified.
Abstract: A surprising functional association between TRPM3, a mysterious member of the family of transient receptor potential (TRP) cation channels, and the sulphated version of pregnenolone, 'mother' of all steroid hormones, has been identified.
TL;DR: Results suggest that endogenous estradiol formation is pivotal for SH-SY5Y cell viability, and selective targeting of neurosteroidogenic pathways may constitute an interesting strategy against H(2)O(2)-evoked neurodegenerative processes.
TL;DR: This first report of a selective direct enhancement of synaptosomal dopamine release by PS at picomolar concentrations via an NMDAR dependent mechanism raises the possibility that dopaminergic axon terminals may be a site of action for this neurosteroid.
Abstract: Pregnenolone sulfate (PS) is a neuroactive steroid that directly modulates glutamate and GABAA receptor function, suggesting that it may regulate the balance between excitatory and inhibitory neurotransmission (Farb and Gibbs 1996; Gibbs and Farb 2000). PS also modulates the activity of AMPA and kainate receptors (Wu et al. 1991), σ receptors (Monnet et al. 1995; Hayashi et al. 2000), and certain voltage-gated calcium channels (Bukusoglu and Sarlak 1996; Hige et al. 2006).
Modulation of synaptic transmission by PS has been demonstrated in multiple experimental paradigms. PS potentiates spontaneously occurring excitatory postsynaptic currents (EPSCs) in hippocampal cell cultures (Park-Chung et al. 1997; Meyer et al. 2002) and in slices prepared from rat pre-limbic cortex (Dong et al. 2005), as well as evoked EPSCs from hippocampal (Schiess et al. 2006) and calyx of Held synapses (Hige et al. 2006). PS also augments NMDAR independent long term potentiation in the rat hippocampus via modulation of L-type Ca2+ channels and σ receptors (Sabeti et al. 2007), and a PS-like retrograde modulatory factor plays a role in plasticity of immature hippocampal synapses (Mameli et al. 2005).
Enzymes for synthesis of pregnenolone from cholesterol (cytochrome P450 scc) and sulfation of pregnenolone to PS (neurosteroid sulfotransferase ST2A1, SULT2B1a) are present in neural tissue (Hojo et al. 2004; Kohjitani et al. 2006). Pregnenolone is inactive at both glutamate and GABAA receptors, indicating that the negatively charged sulfate group of PS is essential for its modulatory activity. Whereas pregnenolone is neutral and lipophilic, permitting rapid permeation across cell membranes, sulfation to form PS results in a negatively charged steroid that could be compartmentalized intracellularly. Pregnenolone sulfotransferase is present in rat C6 glioma cells, where its activity is regulated by AMPA receptors (Kohjitani et al. 2008). Steroid sulfatases are present in rodent, bovine, monkey, and human brain and offer a potential mechanism for inactivation of PS (Compagnone et al. 1997; Mellon et al. 2001; Plassart-Schiess and Baulieu 2001). Collectively, these observations indicate that PS satisfies several classical criteria for identification as a neurotransmitter or neuromodulator: it is synthesized in nervous tissue, has specific receptor pharmacology, and is inactivated by removal of the sulfate group, but the role of PS within the nervous system remains unresolved.
Whether PS is stored and released at physiologically active concentrations by either neurons or glia has remained a controversial issue. The average tissue level of PS in extracts of postmortem aged human brain was found to be 2.8 nM in frontal cortex and 4.6 nM in cerebellum, while blood plasma contains about 380 nM free sulfated steroid. In rat, however, the average level of PS in adrenal tissue was 14 nM but was only 0.64 nM in two of five samples from anterior brain (limit of detection: 0.38 nM) and undetectable in other brain regions (Liere et al. 2004). Another study (Ebner et al. 2006) failed to detect PS in extracts of pooled whole rat brain, with a reported detection limit of 141 pM. Nevertheless, the inability to detect gross tissue levels of PS in various regions of rat brain does not exclude the possibility that significant amounts of this steroid might be present on a cellular level (Schumacher et al. 2008).
Infusion of 10 nM PS into rat striatum via reverse microdialysis results in a significant increase in dopamine (DA) overflow in the striatum that involves NMDA receptor (NMDAR) function, but not σ receptor function (Sadri-Vakili et al. 2008; Gibbs et al. 2006). To ask whether this high affinity effect of PS was exerted via modulation of presynaptic NMDARs located on dopaminergic terminals, we investigated the effect of PS on DA release ex vivo from a preparation of striatal nerve terminals comprised of mixed synaptosomes and synaptoneurosomes (SSNs). We report that PS at concentrations as low as 25 pM induces [3H]DA release from striatal SSNs, while pregnenolone is without effect, suggesting that subnanomolar concentrations of PS could modulate nigro-striatal DA release. To our knowledge, this study is the first to report direct neurosteroid enhancement of NMDAR-dependent synaptosomal transmitter release at subnanomolar concentrations of steroid.
TL;DR: The results indicate the presence of Delta(4) and another derivative pathway in the amphioxus ovary and suggest that the synthesis of sex steroids, particularly estrogen synthesis, may be low in females after spawning behavior.
Abstract: Sex steroids are essential hormones for reproduction in vertebrates. The existence of a sex steroidogenic pathway in invertebrates is controversial because cytochrome P450 (CYP) genes have not been detected in the genomes of an echinoderm and a urochordate. However, cloning and gene expressions of sex steroid-metabolizing enzymes have been reported in a cephalochordate. In this study, in vitro conversion of sex steroids from pregnenolone (P5) to progesterone (P4), from 17a-hydroxyprogesterone (17a-P4) to 17a ,2 0b-dihydroxy-4-pregnen-3-one (17a ,2 0b-P) and 17a, 20a-P, and from androstenedione (AD) to estrone (E1), estradiol-17b (E2), and testosterone (T) were confirmed by an incubation experiment performed using 14 C-labeled precursors and mature ovarian homogenates of the amphioxus Branchiostoma belcheri. In amphioxus, the ability of immature ovaries to synthesize sex steroids was much lower than that of mature ovaries. Post-spawning, the mRNA of CYP11A significantly decreased in females. Transcripts of the CYP19 gene also declined in one-fourth of the females after spawning, although this trend was not supported statistically. The mRNAs of CYP17 and 17b-HSD did not change before and after spawning. Our results indicate the presence of D 4 and another derivative pathway in the amphioxus ovary and suggest that the synthesis of sex steroids, particularly estrogen synthesis, may be low in females after spawning behavior. J. Exp. Zool. 309A:83-93, 2008. r 2008 Wiley-Liss, Inc.
TL;DR: Investigation of the features of AβP-induced changes in intracellular Ca2+ level in primary cultured rat hippocampal neurons using a multisite Ca2-imaging system with fura-2 as a fluorescent probe found that the preadministration of neurosteroids including dehydroepiandrosterone, dehydroepsiandresterone sulfate, and pregnenolone significantly inhibits the increase in intrACEllular calcium level induced by Aβ P.
Abstract: The neurotoxicity of beta-amyloid protein (AbetaP) is implicated in the etiology of Alzheimer's disease We previously have demonstrated that AbetaP forms Ca(2+)-permeable pores on neuronal membranes, causes a marked increase in intracellular calcium level, and leads to neuronal death Here, we investigated in detail the features of AbetaP-induced changes in intracellular Ca(2+) level in primary cultured rat hippocampal neurons using a multisite Ca(2+)-imaging system with fura-2 as a fluorescent probe Only a small fraction of short-term cultured hippocampal neurons (ca 1 week in vitro) exhibited changes in intracellular Ca(2+) level after AbetaP exposure However, AbetaP caused an acute increase in intracellular Ca(2+) level in long-term cultured neurons (ca 1 month in vitro) The responses to AbetaP were highly heterogeneous, and immunohistochemical analysis using an antibody to AbetaP revealed that AbetaP is deposited on some but not all neurons Considering that the disruption of Ca(2+) homeostasis is the primary event in AbetaP neurotoxicity, substances that protect neurons from an AbetaP-induced intracellular Ca(2+) level increase may be candidates as therapeutic drugs for Alzheimer's disease In line with the search for such protective substances, we found that the preadministration of neurosteroids including dehydroepiandrosterone, dehydroepiandrosterone sulfate, and pregnenolone significantly inhibits the increase in intracellular calcium level induced by AbetaP Our results suggest the possible significance of neurosteroids, whose levels are reduced in the elderly, in preventing AbetaP neurotoxicity
TL;DR: In this paper, the effect of cholesterol sulfate (CS) on the expression of progesterone-related genes in KGN cells derived from human granulosa-like tumor was investigated.
Abstract: Cholesterol sulfate (CS) is a component of cell membranes that plays a role in stabilizing the cell membrane. We previously reported that CS increased in the endometrium during implantation, suggesting that CS plays an important role in reproduction. It has been reported that CS regulates progesterone and pregnenolone production in the placenta, adrenal glands and ovary. The regulatory mechanisms of steroid hormone production by CS, however, are still unknown. In the present study, we investigated the effect of CS on the expression of progesterone production-related genes in KGN cells, derived from human granulosa-like tumor. KGN cells were cultured with CS (10 muM) or cholesterol (10 muM) in the presence of 8-bromo-cAMP (1 mM). Progesterone levels in the culture media were measured by enzyme linked fluorescent assay at 24 h after treatment of CS and cAMP. Total RNAs were extracted for quantitative real time RT-PCR with specific primer of StAR protein, P450scc, HSD3B2, ferredoxin and ferredoxin reductase. Whole cell lysates were extracted for western blot analysis with antibody for StAR protein. Progesterone concentration in the culture medium increased to 38-fold by treatment of cAMP. CS significantly reduced progesterone concentration by 30% compared with those of cAMP treatment (p<0.05), while cholesterol did not change the progesterone concentration. CS treatment down-regulated the expression of StAR mRNA and P450scc mRNA was to 54% and 60%, respectively (p<0.05). Western blot analysis revealed that the amount of StAR protein was also reduced by CS treatment. The expression of HSD3B2 mRNA was up-regulated to 3.4-fold by treatment of cAMP. The expression of ferredoxin and ferredoxin reductase mRNA was not affected by CS treatment. These data implied that CS has an inhibitory effect on progesterone production by regulating the expression of StAR and P450scc gene expression.
TL;DR: Plasma PREG-S and deoxycorticosterone levels are differentially regulated by HPA axis modulation in human plasma and alcohol-dependent patients show a blunted PREg-S response to adrenal stimulation and a delayed deoxyCortic testosterone response to oCRH challenge.
TL;DR: Evidence is provided for Schwann cells as a major producer of steroid hormones and pregnenolone production by P450scc as an important regulatory step during myelination.
Abstract: To investigate the production of steroid hormones by Schwann cells and to examine the regulation of steroid hormone production during myelination, cultures of rat Schwann cells were differentiated into their myelinating phenotype in the absence of neurons with dibutyryl cAMP (db-cAMP). During this process, the expression of P450scc (involved in steroid biosynthesis) was elevated at both the mRNA and protein levels as evident in RT-PCR, Western blots, and immunostaining. Labeling of the cells with [14C] acetate revealed enhanced production of pregnenolone during differentiation into the myelinating phenotype. Disruption of P450scc’s activity with an inhibitor diminished the extent of differentiation into the myelinating phenotype as levels of mRNA and protein expression of myelin protein zero (P0) declined. However, the effect was reversed with the addition of pregnenolone. Furthermore, when the differentiating cultures were treated with pregnenolone, mRNA expression of P0 was upregulated, suggesting the stimulation of the differentiation process. Together, these results provide evidence for Schwann cells as a major producer of steroid hormones and pregnenolone production by P450scc as an important regulatory step during myelination.
TL;DR: Data suggest that, in H295R cells, Hint2 is required for an optimal steroidogenic response, possibly because of a particular signalling function exerted within the mitochondria and that still remains to determine at the molecular level.
Abstract: Hint2 belongs to the superfamily of histidine triad hydrolase enzymes. Recently, it has been shown to influence the mitochondria-dependent apoptosis occurring in hepatocytes, but its mechanism of action is still obscure. Here, we demonstrate that Hint2 is expressed in the mitochondria of H295R cells and in normal adrenals, and that this protein is involved in steroidogenesis. The presence of Hint2 in H295R cells was revealed by RT-PCR and by immunoblot analysis of subcellular fractions. The protein appeared associated with mitochondrial membranes, probably facing the interior of the organelle. Hint2 overexpression in H295R cells had no effect on pregnenolone secretion elicited by angiotensin II or K+, whereas protein silencing with specific small interfering RNA resulted in a marked reduction of the steroidogenic response. The duration of the mitochondrial calcium signal induced by angiotensin II was also reduced upon Hint2 down-regulation with small interfering RNA, but not affected after its overexpression, suggesting that under basal conditions, Hint2 is optimally expressed, and not rate limiting in steroidogenesis. Moreover, Hint2 also appeared involved in Ca2+-independent pathways leading to steroid formation. Indeed, pregnenolone formation in response to either forskolin or a hydroxyl analog of cholesterol was markedly reduced after Hint2 silencing. Calcium-dependent and calcium-independent actions of Hint2 on steroidogenesis could be related to its ability to maintain a favorable mitochondrial potential. In conclusion, these data suggest that, in H295R cells, Hint2 is required for an optimal steroidogenic response, possibly because of a particular signalling function exerted within the mitochondria and that still remains to determine at the molecular level.
01 Jan 2008
TL;DR: Investigation of expression of the steroid sulfotransferase SULT2B1a, which catalyzes PREGS production, is inhibited by activation of excitatory amino acid receptors of the AMPA subtype, via facilitation of intracellular NO signaling.
TL;DR: The results of this study indicate that increased cholesterol in the kainate lesioned hippocampus is mostly metabolized to oxysterols, and not neurosteroids.
Abstract: Our previous study showed an increase in total cholesterol level of the hippocampus after kainate-induced injury, but whether this is further metabolized to neurosteroids is not known. The first step in neurosteroid biosynthesis is the conversion of cholesterol to pregnenolone by the enzyme cytochrome P450 side chain cleavage (P450scc). This study was carried out to elucidate the expression of this enzyme in the kainate-lesioned rat hippocampus. A net decrease in P450scc protein was detected in hippocampal homogenates by Western blots at 2 weeks post-kainate injection (time of peak cholesterol concentration after kainate injury). Immunohistochemistry showed decreased labeling of the enzyme in neurons, but increased expression in a small number of astrocytes. The level of pregnenolone was also analyzed using a newly developed gas chromatography-mass spectrometry (GC-MS) method, optimized for the rat hippocampus. A non-significant tendency to a decrease in pregnenolone level was detected 2 weeks post-lesion. This is in contrast to a large increase in oxysterols in the lesioned hippocampus at this time (He et al. 2006). Together, they indicate that increased cholesterol in the kainate lesioned hippocampus is mostly metabolized to oxysterols, and not neurosteroids.